Projets de recherche

Code: 05D08LM

Violations for the steroid testosterone are typically the second most common (after Cannabis) of all banned substances. To detect testosterone abuse an initial screening of athletes’ urine occurs and the testosterone/epitestosterone ratio is measured. Enhanced levels of testosterone with respect to its very close analogue epitestosterone are indicative of steroid abuse. The new 2005 WADA Prohibited Substances List took effect on January 2005 and changed the level of the testosterone/epitestosterone ratio at which further investigation for potential testosterone abuse must be conducted from 6/1 to the lower level of 4/1. The measurement of T/E ratios is technically not a trivial issue ant his reduction in ratio makes it even more essential that WADA-accredited laboratories can accurately determine T/E ratios. A main aim of this project is the production and certification of such a urine matrix certified reference material (CRM) certified to a very accurate level for the concentrations of both testosterone and epitestosterone (and their ratio) will also be produced to provide laboratories with a suite of reference materials to ensure that as many parts of their analytical process as possible can be controlled. These certified reference materials will be produced by Australia’s National Measurement Institute (NMI) using techniques which are considered to be primary methods to ensure the highest accuracy and lowest uncertainty. Once they are produced these reference materials can be used by all WADA accredited laboratories to test their specific methods and ensure accurate and internationally comparable results are produced. These materials will provide an unequivocal international benchmark for all measurements of these steroids around the world.

Main Findings

The preparation and characterisation of two matrix reference materials to underpin the accuracy of measurements of testosterone to epitestosterone (T/E) ratios has been successfully completed. A methanol solution of testosterone and epitestosterone was prepared as reference material NMIA MX006 and distributed into 2386 glass ampoules, each containing 1 mL. Composite human urine containing natural levels of epitestosterone glucuronide and testosterone glucuronide, the key excreted metabolites of testosterone and epitestosterone, was fortified with additional testosterone glucuronide to an approximate T/E ratio of 4. This material (designated NMIA MX005) was distributed into 1282 bottles in 20 mL aliquots and freeze-dried to maximise stability in storage. During the first two years of the project, high accuracy methods of analysis for testosterone, epitestosterone and their glucuronides were developed, optimised and validated. Pilot batches of the two proposed reference materials were produced to demonstrate the feasibility of preparing homogenous and stable products with the required property values. This enabled successful preparation of bulk quantities of the candidate certified reference materials (CRM), which were then apportioned into individual units. These were then extensively evaluated to provide initial reference values and associated uncertainties during the second year of the project. Assessment of homogeneity and stability of the CRMs during storage, transport and use was performed and alternate high accuracy methods of analysis developed to investigate possible measurement biases. Tasks performed during the third and final year of the project involved completing the estimates of the uncertainty associated with the reference values for the measurands to be certified. These measurands are the mass fractions and concentrations of testosterone and epitestosterone in the methanol solution (NMIA MX006), the mass fractions and concentrations of the glucuronide conjugates of testosterone (as testosterone) and epitestosterone (as epitestosterone) following reconstitution of the freeze dried urine (NMIA MX005), and the T/E ratios in both materials. The long term stability trials on both reference materials at their normal storage temperature (-20oC) continued and no significant changes were observed in the measurands.

Code: 05B2MT 

Insulin-like growth factors (IGFs) are circulating peptides that are involved in the regulation of cell proliferation, differentiation and apoptosis. In particular IGF-I has been found to be a critical modulator of skeletal muscle growth when administered locally rather than systemically. The infusion of IGF-I into target tissues such as selected skeletal muscles results in a significant increase in total protein and DNA content, an effect that is highly desirable for athletic performance in various sports disciplines. Owing to the growth promoting properties of IGF-I it belongs to the prohibited list of the World Anti-Doping Agency, and the determinations of its abuse or corresponding synthetic derivatives is of paramount importance. The amino acid composition of recombinant IGF-I is identical to that of endogenously produced IGF-I, and preliminary approaches to reveal IGF-I misuse are based on its quantification. By means of mass spectrometry, a precise determination of IGF-I amounts in human plasma is aspired in addition to an unambiguous identification of this peptide. Moreover, synthetic analogues such as R3 -IGF-I and long IGF-I are commercially available, and strategies enabling the detection of three structurally related compounds are required. A selective isolation of IHG-I and analogues from human plasma is possible by means of immunoaffinity extraction and subsequent liquid chromatography coupled to mass spectrometry provides substantial data allowing a qualitative and quantitative determination of target analyses.

Main Findings: 

Human IGF-1 is a promising part of strategy to reveal the abuse of human growth hormone by serving as a biomarker. Therefore, it is of paramount importance to quantify the plasma level of IGF-1 in order to be able to compare the results with valid reference ranges and thus to distinguish between 'normal' and 'abnormal' concentrations. The definition of such reference values is still ongoing because there are many parameters to consider. Regarding the IGF-1 analogues as a possible threat in sports and doping control analysis, there is a lack of information because pharmacological data in human are currently not avaible. Recent investigations in the internet have, however, shown that LONG(tm)R(3)IGF-1 in particular is already in use. Althought it is not approved for the use in humans, it is probably favoured due to its increased potency (e.g. prolonged half-life). Based on its higher biological activity the administered doses and, hence the costs, are much lower than excepted for an application of recombinant human IGF-1. Thus, an assay enabling the determination in athletes' plasma specimens is required. The described procedure allows the validquantification of human IGF-1 as well as the unequivocal identification of its analogues LONG(TM)R(3)IGF-1 and des(1-3) IGF-1. The combination of immunoaffinity isolation and purification with LC-ESI-MS/MS provides utmost specifity for the analysis of banned peptides as recently demonstrated. It enables absolute quantification without distortion caused by, e.g., cross-reactions.

Code: 05D4JY

Mesocarb (Sydnocarb®) is an amphetamine-related psychostimulant that have been used for doping purposes due to their central nervous system stimulating effects. Moreover, it is very likely that mesocarb is being used by drug addicts. Mesocarb is included in the World Anti-Doping Agency’s list of substances and methods that are prohibited in sports. Mesocarb is chemically a cyclic and mesoionic N-phenylcarbamoyl-3-(betaphenylisopropyl) sydnone imine. The dominant urinary metabolites of mesocarb are identified to be its mono-, di- and trihydroxylated isomers. The exact chemical structures of regioisomeric metabolites of mesocarb have not been solved yet. Furthermore, the metabolites are not commercially available. According to the WADA’s international laboratory standard and ISO 17025 standard, well characterized pure reference materials are recommended to be used as references in the analysis. In this project a high-yielding and selective synthesis methods for the preparation of mesocarb metabolites as reference substances will be developed. The synthesized metabolites will be purified and characterized and their chemical stability will be tested. The synthesized substances will be used to solve so far unknown regiochemical structures of the main urinary metabolites of mesocarb. Using the synthesized substances, the feasibility of various screening methods used in doping laboratories for the abuse of mesocarb will be tested, and a liquid chromatographic – tandem mass spectrometric confirmation method will be developed and validated. Finally, the synthesized mesocarb metabolites would be available free of charge as reference substances to the world’s antidoping community. The pure and fully characterized reference substances would enable the reliable and legally defensible analysis of mesocarb and could also be used in quality assurance and in development of new analytical methods.

Main Findings: 

Mesocarb (Sydnocarb®) is an N-alkylated amphetamine derivative, and has been used for doping purposes due to its central nervous system stimulating effects. It belongs to the list of prohibited substances and methods in sports of World Anti-Doping Agency (WADA). According to the WADA’s international standard for laboratories and ISO 17025 standard, well-characterized reference compounds should be used for the identification of a prohibited substance, if available. Presently, the metabolites of mesocarb are not commercially available, and additionally, the exact chemical structures of several mesocarb metabolites have not been solved yet. In this WADA-funded research project we synthesized six potential mono-, di-, and trihydroxylated regioisomeric metabolites of mesocarb for the doping analytical purposes. The synthesized metabolites were purified, characterized and compared with the in vitro synthesized mesocarb metabolites using human liver enzymes and with the in vivo formed metabolites extracted from human urine after oral administration of mesocarb. An LC-MS/MS-method for the characterization of mesocarb and its metabolites in urine was developed. p-Hydroxymesocarb was found to be the main (conjugated and non-conjugated) metabolite in human urine, emphasizing the importance of its synthesis and availability as a reference material with respect to doping analysis. The synthesized and fully characterized p hydroxymesocarb enables the reliable and legally defensible confirmation analysis of mesocarb, and could also be used in quality assurance and in development of new analytical methods. The metabolite synthesized within this project will be available without charge to all WADAaccredited anti-doping laboratories.

Code: 05B9RK 

It is known that anabolic steroids are very effective in promoting muscle growth. The detection of synthetic anabolic steroids is now very effectively carried out by WADA laboratories but the effective detection of the abuse of naturally occurring anabolic steroids is still a major challenge. Doping control laboratories need criteria that allow endogenous steroids to be distinguished from their synthetic analogues in urine. For some endogenous steroids such as testosterone the criteria have been established and measurements of the T/E ratio coupled with isotope ratio measurements can effectively detect such doping. There are however several other endogenous androgenic steroids which can potentially increase active androgen levels and boost performance. 4-androstenediol and 5-androstenediol are two such prohormones which are banned but for which there is incomplete understanding of their metabolism. Recent evidence indicates that at least in some individuals the administration of 4-androstenediol does not lead to a significant change in the T/E ratio. Thus there is a need for improved detection capabilities which can not only detect the abuse of endogenous androgens but also specify which steroid has been administered. It is proposed to use the same methodology which has been developed in this laboratory for the detection of dehydoepiandrosterone (DHEA) abuse. Specific markers arising from 4-androstenediol and 5- androstenediol abuse will be looked for in both the glucuronide and sulfate steroid fractions. Once identified these compounds will be used both for GCMS screening and for GC-C-IRMS confirmation procedures.

Main Findings: 

Compound specific detection (CSD) of endogenous steroid abuse using complementary Gas Chromatography-Mass Spectrometry (GC-MS) and Gas Chromatography-Combustion-Isotope Ratio Mass Spectrometry (GC-C-IRMS) techniques is an important requirement for effective doping control in sport. This project was undertaken to determine if 4-androstenediol (4-ADIOL; androst-4-ene- 3β,17β-diol) and 5-androstenediol (5-ADIOL; androst-5-ene-3β,17β-diol) administration could be selectively identified in urine. Single and multiple oral doses of 4- and 5-ADIOL were performed on two volunteers with human ethics approval and informed consent to monitor changes in urinary endogenous steroid excretion and 13C content. The results showed significant increases in the excretion of androsterone glucuronide and etiocholanolone glucuronide from 4- and 5-ADIOL administration. This behaviour, which is similar to that observed for dehydroepiandrosterone and androstenedione in previous studies, is an effective screening indicator of endogenous steroid administration. 4-ADIOL was found to produce the diagnostic urinary dehydrogenation products: androst-2,4-diene-17-one and androst-3,5-diene- 17-one. These can be used as screening markers in the GC-MS steroid profile to identify suspicious samples that require confirmation by carbon isotope ratio (δ13C) analysis using GC-C-IRMS. Differences in post-administration detection times based on Δδ13C values, relative to δ13C measurement of an endogenous reference compound, were found between glucuronide and sulfoconjugated steroids that were dependent on the individual’s metabolism. Preferential glucuronide excretors provided a detection period of up to 72 hours, while sulfoconjugate excretors may provide up to 68 hours. The administration of 5-ADIOL did not result in the urinary excretion of the diagnostic diene marker compounds, thereby enabling a distinction to be made between the 4- and 5-ADIOLs. Additionally, CSD between 4- and 5-ADIOL may be performed using δ13C analysis of the etiocholanolone sulfoconjugate relative to the sulfoconjugates of androsterone, epiandrosterone and dehydroepiandrosterone. The detection of 5-ADIOL doping based on Δδ13C values, derived from sulfoconjugates greater than 4.0‰ is capable of achieving a 60 hour post-administration time.

Code: R04A12PS

The aim of the project is to evaluate a procedure to detect the use of any substance (i.e. erythropoietin and analogs) or method (i.e. homologous or autologous transfusions) that induce an increase in total haemoglobin.

The proof of principle is based in monitoring variations in the oxygen saturation curve by comparing measurements under normoxia or hypoxia in the resting state. It is proposed that the results will show that individuals who resorted to any of the aforementioned prohibited substances or methods will show a better adaptation under hypoxic conditions, evidenced by a significantly increased oxygen saturation value.

In all, the overall objective is to develop an indirect field test to instantly measure this type of doping in addition to urinary and/or blood tests used to that effect. It is envisioned to use such method on the same grounds as the currently utilized hematocrit determination.

Main findings

The aim of this study was to investigate an indirect method based on variations in haematological markers that could be used to identify haemoglobin-enhancing substances or methods that improve oxygen transfer.

The selection of statistical markers has been realised from experimental variations obtained during different phases including an increase in training volume at sea level, high altitude training, blood withdrawal and autologous blood transfusion. Blood arterial oxygen saturation under hypoxia conditions at resting state, like other markers, could not be taken into account due to the absence of statistical uniformity. The markers selected were hematocrit (Hct), haemoglobin concentration ([Hb]) and stimulation index (Off-hr). An absolute norm of variation (normΔ) for each selected markers allowing the distinction between normal and abnormal variations was established at the time. The absolute norms of variation obtained are:

normΔHct>6%

normΔ[Hb]>4%

normΔOff-hr>20%

Application of this method for one day competitive trials would ideally consist in taking the first blood sample 15 days before the competition and the second one upon its completion. For competitive trials of several days or weeks or during championships lasting several months blood samples should be taken without warning at a maximum interval of 15 days. In the future, this maximal interval could be increased with use of localisation data collected with the ADAMS system, and specifically those linked with presence at high altitude or not of an athlete.

Code: 04D22CG

The aims of the proposed research study are the following:

• Unification of the WADA Accredited Doping Control Laboratories screening procedures of classes of prohibited substances including classical small molecules like stimulants, narcotics, steroid agents, diuretics, etc, in order to free laboratory resources to new classes of prohibited substances: e.g. proteins. Modern sensitive high mass resolution, full mass range fast scanning mass spectrometer technology Time-Of-Flight, coupled with GC and LC chromatographic systems, capable to cover all classes of small molecules prohibited substances in minimum analytical runs per samples will be used. Sample preparation techniques using the minimum of steps but compatible with all classes of small molecules/prohibited substances will also be used. Incorporation of new drugs or metabolites, belonging to similar classes of molecules, with minimum or no changes to the laboratory preparative/analytical methods will be facilitated.

• Development of two new analytical tools, based on the instrumentation referred to in the previous paragraph, in order to widen the spectrum of the detection of prohibited substances or metabolites that do not exist as reference materials in the laboratory: a) creation of predictive analytical data, for molecules that are computer designed in the laboratory, b) bioassay-directed identification of unknown molecules

Main findings

A novel, rapid and easy-to-use method, EPO WGA MAIIA, for determination of aberrant EPO isoform subpopulations in urine or serum, has been tested for its use as an EPO doping control method. The method separates EPO subpopulations due to their different interactions with the lectin wheat germ agglutinin (WGA). The glycosylated structures on recombinant epoetins show stronger interaction with the lectin, probably due to their higher content of polylactose amine. The WGA-based separation of isoforms and the subsequent ultrasensitive EPO determination is rapidly carried out within a few square cm of a thin porous layer formed as a test strip, using an image scanner for quantification. The test takes only 20 min. to perform and is well suited both for determination of single samples and for large series. Before analysis with the EPO WGA MAIIA method, EPO is purified and concentrated from urine or plasma by use of a newly developed disposable EPO affinity purification device (www.maiiadiagnostics.com). With this easy-to-use sample preparation device EPO can rapidly be captured from large sample volumes and be eluted in a final volume of only 55 µL. The high EPO recovery of 65%, and the retained isoform distribution, makes the device a useful pre-step tool also for e.g. IEF, SDS-PAGE and LC/MS. The EPO WGA MAIIA method allows detection of recombinant EPO in urine specimens from patients up to about 7 days after the last injection (p<0.0001). Recombinant epoetin e.g. alpha, beta, omega, delta, zeta and four Chinese types (p<0.0001), and EPO analogues like Aranesp (p<0.0001) and Mircera can be distinguished from endogenous EPO isoforms. Mircera shows less interaction with WGA compared to endogenous EPO, while recombinant EPO:s show stronger and Aranesp shows the strongest interaction. Only 2 pg of EPO is required for isoform detection, which is about 1/10 of the amount of EPO required for the presently used IEF based doping method. When rhEPO beta and endogenous EPO appear in the same sample it is possible to detect rhEPO down to a level where it constitutes only 40% of total EPO. Besides measuring the interaction of the various types of EPO with WGA, it is possible in the same test strip to utilize also their interaction with the anti-EPO immobilized in the detection zone (see J. Immunol. Meth. 339 (2008) 236–244). By interpreting the antibody interaction profile, using the EPO AbQ MAIIA algorithm, it is possible to distinguish EPO and epoetins from EPO analogues like Aranesp and Mircera. The EPO WGA MAIIA test gives also an estimate of the EPO concentration in the eluate, enabling calculation of optimal application volume for the IEF or SDS-PAGE confirmation test. The recommended test set-up for doping control utilizes EPO WGA MAIIA for identification of epoetins and Aranesp, while Mircera preferably is identified by EPO AbQ MAIIA. The EPO WGA MAIIA test classifies all tested epoetin varieties and EPO analogues correctly, shows good resolution between endogenous EPO and epoetins, and can measure very low amounts of EPO. The quality controlled reagents will be supplied worldwide as a complete kit. The hands-on time is reduced compared to presently accredited tests, which significantly decreases the analysis cost. The excellent results and the easyto-use concept seem to fulfil the requirements for a screening EPO doping control test. Such a test makes it possible to considerable increase the number of EPO doping controls performed without increasing the total analysis cost.

Code: T04D27JD

The aim of this project is to set criteria for the misuse of glucocorticosteroids including synthetic glucocorticosteroids, natural glucocorticosteroids (cortisone/hydrocortisone) and Synacthen as stimulators of the adrenal cortex. For synthetic glucocorticosteroids, a general methodology has to be established including oral and intra-muscular administration as systemic routes and nasal and/or inhalation administration as local routes, with a high probability of systemic effect. The other local routes are to be considered as having a low probability of systemic action. According to this scheme, a two step study was built. First, for each test substance, time-related studies will be carried out under well controlled clinical conditions (ie clinical trials) for the systemic routes (oral and IM), as well as for the local routes with a high probability of systemic recovery (nasal and/or inhalation). These results will be used to set preliminary criteria for positivity (part I). Secondly, for some substances, time-related studies will be performed under ambulatory treatments (uncontrolled conditions) for the other local routes (dermal, intra-articular). These results will be used to confirm the preliminary criteria or to modulate them if necessary (part II). Moreover, urine indicators of native cortisol metabolism breakdown will be monitored by GC-MS in parallel to the measure of synthetic glucocorticosteroids urinary concentration by LC-MS/MS, whatever the mode of administration (part I and part II of this study). It is expected that these additional results will offer a way to confirm the general authorization for local routes or to modulate their initial status, especially if there is any evidence of a substance-related health risk due to cortico-adrenal gland suppression. For natural glucocorticosteroids (cortisone/hydrocortisone) and Synacthen, the determination of endogenous glucocorticosteroid profiles by GC-MS analysis is also regarded as a key step in this study. Consequently, complementary analyses of the main cortisol metabolites by GC-C-IRMS and of other native steroid compounds by CC-MS will be performed.

Main Findings:

AFLD Laboratory: Via deux études de population distinctes nous avons établi des seuils de suspicion correspondant à la moyenne + 2 écarts types, puis des seuils de positivité correspondant à une déplétion isotopique de 3 ‰ sur la base de la moyenne +é- 3 écarts types. Ces seuils sont d’accord avec les études d’excrétion au cortisol et à la cortisone. Ces études contrôles ont démontrée que l’interprétation des rapport Y+⁄THF et THS et F⁄THS lorsqu’ils sont pris ensemble sont globalement en concordance avec la technique. Nos études étant limitées a des sous population d’origine caucasienne, l’universalité des résultats devrait être examinée a travers des études de sus population multiethnique. Une variante de la méthode directe d’oxydation utilisée pour IRMS avec séparation des métabolites du cortisol et de la cortisone de la désoxycortocosterone avant l’oxydation améliorait la spécificité de l’analyse isotopique. Lausanne laboratory : Les résultats de dosage obtenus après investigations des divers glucocorticostéroïdes montrent une grande variabilité en fonction des modes d’administration, des concentrations maximales éliminées par les individus, du rapport entre les fractions libres et conjuguées, de la durée d’élimination. Néanmoins, au vu des résultats, il est déjà envisageable de distinguer les voies systémique (orale, intramusculaire) des autres voies locales d’administration (pulmonaire, nasale) en fonction des concentrations urinaires éliminées des divers glucocorticostéroïdes investigués et de leur rémanence dans l’urine. Néanmoins, les voie intra-articulaire et péridurale devront être re-évaluées car ne peuvent être différenciées des voies systémiques. Après synthèse des résultats obtenus par les laboratoires de Paris, Lausanne et Sydney, il sera probablement possible d’affiner le seuil de positivité, permettant de réglementer l’administration des glucocorticostéroïdes. D’autre part, il serait intéressant de poursuivre et d’approfondir les investigations au niveau des injections intra-articulaires et péridurales, et éventuellement des autres modes d’administration (péri-articulaire, péri-tendineux, mais aussi les pommades, collyres et autres solutions). Au vu des résultats préliminaires, il serait possible en dosant le Cortisol de distinguer la voie systémique (orale et surtout intramusculaire) des autres voies locales d’administration, comme l’inhalation pulmonaire. Un affaissement du Cortisol urinaire avec des concentrations proches de zéro pourrait indiquer la nature de l’administration du glucocorticostéroïde. Néanmoins, des analyses complémentaires devraient encore être réalisées dans le but de confirmer et de généraliser cette observation. En particulier, sur un plus grand nombre de volontaires et surtout sur les échantillons provenant des IA et des autres applications locales.

Code: 04D06DH

The recent identification of two designer androgens, norbolethone and tetrahydrogestrinone (THG), has raised new concerns regarding risks from novel designer androgens custom-produced as undetectable sports doping agents. These two synthetic androgens were produced by modification of existing steroids to produce previously unknown (THG) or non-marketed (norbolethone) potent androgens. THG was produced from gestrinone, a progestin known to have androgenic activity and banned by WADA. Yet, among 36 commercially marketed progestins many have similar or greater androgenic potential than gestrinone and the capability to be chemically modified, potentially forming potent and currently undetectable designer androgens. Banning all synthetic progestins is not feasible given their importance in female reproductive health. Therefore it is essential to characterize the available progestins according to their risk profile and to develop detection methods for those with most androgenic activity and most capable of being developed into so-far undetectable doping agents. This project involves 3 stages, the first involves an in vitro yeast based androgen bioassay that we used to provide the first proof that THG was a potent androgen. This in vitro androgen bioassay is a sensitive and efficient screening method that can be applied to all available progestins and their analogs or metabolites as compared with a reference panel of synthetic androgens. The second stage involves confirming the androgenic bioactivity identified as positive in the in vitro screening bioassay by an in vivo mouse-based androgen bioassay. The third stage involves developing urinary Mass Spectrometric detection methods for progestins with significant in vitro and in vivo androgenic bioactivity.

Main Findings

A comprehensive review of commercially marketed synthetic progestins was completed using the yeast-based in vitro androgen bioassay. This led to a detailed description of steroid androgen receptor (SAR) for androgenic activity of synthetic progestins. The practical implications of this analysis for the WADA antidoping program were that the risk from newer synthetic progestins was relatively low as they lacked significant intrinsic androgenic potency. The major risk was from the older, first generation synthetic progestins, especially those derived from 19-nortestosterone, which had significant intrinsic androgenic potency that could be enhanced by simple chemical modifications. Nevertheless existing GC/MS profiling was already available for these compounds. These conclusions are supported by the rarity of novel progestin-derived illicit designer androgens subsequent to the discovery of THG. The simplified and modified mouse in vivo androgen bioassays gave quite consistent findings with rank order of potency of the in vitro androgen bioassays. Specific class-based comparisons confirmed that the newer third generation synthetic progestin, as exemplified by nesterone, had negligible androgenic bioactivity whereas the older early generation progestin, norgestrel, had significant intrinsic androgenic potency in vivo. These findings confirmed that no further detailed metabolic analysis of excretory products of modern progestins in humans is warranted as the significant risks are already covered by the known commercially marketed progestins.

Publications

L. McRobba, D.J. Handelsman, R. Kazlauskas, S. Wilkinson, M.D. McLeode, A.K. Heather. Structure–activity relationships of synthetic progestins in a yeast-based in vitro androgen bioassay. Journal of Steroid Biochemistry & Molecular Biology, 110 (2008) 39–47.

Code: R04D35CA

This project is aimed at providing rapid answers to testing authorities following the seizure and/or the discovery of use of a new steroid. As a matter of fact little can be done at the analytical level until the substance has bee fully characterized, its urinary metabolites identified, the reference material made available and its potential performance-enhancing properties investigated. Since 2000, the testing authorities have been alerted to the use of new steroids by certain athletes. Public comments made by individuals involved in the Balco scandal are adding to the information obtained from other sources, mainly informants, to the effect that other “designer” potent and undetectable steroids have been prepared and would be available to some athletes. That has become a certainty in December 2003, following seizure at the Canadian border of hGH and two steroid products one of which being THG. The identification of the second one, DMT (17α-methyl-5α-androst-2-en-17β-ol) has been done within the scope of this grant application. Lastly, urine samples of athletes at “high risks” have been found to contain metabolites of two isomers of a steroid which are not currently tested for. Steroids or urinary steroid metabolites will be identified employing analytical techniques such as mass spectrometry and NMR; structures will be proposed. The synthesis of reference standards will permit the full characterization of the compounds (parent and metabolites)> The hormonal properties of the steroids, androgenic/anabolic, estrogenic and progestagenic will be assayed. Studies will be conducted to enable the identification of the metabolites (phase I and II) which will permit ultimately their inclusion to testing methods.

Main Findings

We consider having fully met the objectives contained in the original application. New steroids were characterised: DMT1 ‐ 17α‐methyl‐5α‐androst‐2‐en‐17β‐ol major isomer along with the 3‐en isomer, methyldrostanolone ‐ 2α,17α‐dimethyl‐5α‐androstan‐17β‐ol‐3‐one (sold under the names of Superdrol, Methasterone; and Guggulsterone ‐4,17(20)‐pregnadiene‐3,16‐dione. Anticipating what could be the new molecules introduced clandestinely we have synthesized 17‐methylated derivatives of popular steroids such as methenolone and its isomer stenbolone. The phase I metabolites were successfully produced from incubations with cryopreserved human hepatocytes for several steroids particularly drostanolone and its 17‐methylated derivative, “Superdrol”. Structures were proposed for a novel metabolite hydroxylated in C‐2 and confirmed by chemical synthesis. NMR and mass spectrometry were utilised to characterise the metabolites. The model failed however to produce conjugated phase II metabolites and significant amounts of phase 1 metabolites from desoxymethyltestoterone, DMT. In spite of these limitations, the identification of metabolites was much easier in incubation medium when compared to the heavily interfered urine matrix. The utilisation of human hepatocytes significantly reduces to the minimum the administration to human volunteers. We have extended by the work in order to finish the characterisation of the metabolites of methyldrostanolone and drostanolone including new ones that were not reported previously.

Code: T04D27JD

The aim of this project is to set criteria for the misuse of glucocorticosteroids including synthetic glucocorticosteroids, natural glucocorticosteroids (cortisone/hydrocortisone) and Synacthen as stimulators of the adrenal cortex. For synthetic glucocorticosteroids, a general methodology has to be established including oral and intra-muscular administration as systemic routes and nasal and/or inhalation administration as local routes, with a high probability of systemic effect. The other local routes are to be considered as having a low probability of systemic action. According to this scheme, a two step study was built. First, for each test substance, time-related studies will be carried out under well controlled clinical conditions (ie clinical trials) for the systemic routes (oral and IM), as well as for the local routes with a high probability of systemic recovery (nasal and/or inhalation). These results will be used to set preliminary criteria for positivity (part I). Secondly, for some substances, time-related studies will be performed under ambulatory treatments (uncontrolled conditions) for the other local routes (dermal, intra-articular). These results will be used to confirm the preliminary criteria or to modulate them if necessary (part II). Moreover, urine indicators of native cortisol metabolism breakdown will be monitored by GC-MS in parallel to the measure of synthetic glucocorticosteroids urinary concentration by LC-MS/MS, whatever the mode of administration (part I and part II of this study). It is expected that these additional results will offer a way to confirm the general authorization for local routes or to modulate their initial status, especially if there is any evidence of a substance-related health risk due to cortico-adrenal gland suppression. For natural glucocorticosteroids (cortisone/hydrocortisone) and Synacthen, the determination of endogenous glucocorticosteroid profiles by GC-MS analysis is also regarded as a key step in this study. Consequently, complementary analyses of the main cortisol metabolites by GC-C-IRMS and of other native steroid compounds by CC-MS will be performed.

Main Findings:

AFLD Laboratory: Via deux études de population distinctes nous avons établi des seuils de suspicion correspondant à la moyenne + 2 écarts types, puis des seuils de positivité correspondant à une déplétion isotopique de 3 ‰ sur la base de la moyenne +é- 3 écarts types. Ces seuils sont d’accord avec les études d’excrétion au cortisol et à la cortisone. Ces études contrôles ont démontrée que l’interprétation des rapport Y+⁄THF et THS et F⁄THS lorsqu’ils sont pris ensemble sont globalement en concordance avec la technique. Nos études étant limitées a des sous population d’origine caucasienne, l’universalité des résultats devrait être examinée a travers des études de sus population multiethnique. Une variante de la méthode directe d’oxydation utilisée pour IRMS avec séparation des métabolites du cortisol et de la cortisone de la désoxycortocosterone avant l’oxydation améliorait la spécificité de l’analyse isotopique. Lausanne laboratory : Les résultats de dosage obtenus après investigations des divers glucocorticostéroïdes montrent une grande variabilité en fonction des modes d’administration, des concentrations maximales éliminées par les individus, du rapport entre les fractions libres et conjuguées, de la durée d’élimination. Néanmoins, au vu des résultats, il est déjà envisageable de distinguer les voies systémique (orale, intramusculaire) des autres voies locales d’administration (pulmonaire, nasale) en fonction des concentrations urinaires éliminées des divers glucocorticostéroïdes investigués et de leur rémanence dans l’urine. Néanmoins, les voie intra-articulaire et péridurale devront être re-évaluées car ne peuvent être différenciées des voies systémiques. Après synthèse des résultats obtenus par les laboratoires de Paris, Lausanne et Sydney, il sera probablement possible d’affiner le seuil de positivité, permettant de réglementer l’administration des glucocorticostéroïdes. D’autre part, il serait intéressant de poursuivre et d’approfondir les investigations au niveau des injections intra-articulaires et péridurales, et éventuellement des autres modes d’administration (péri-articulaire, péri-tendineux, mais aussi les pommades, collyres et autres solutions). Au vu des résultats préliminaires, il serait possible en dosant le Cortisol de distinguer la voie systémique (orale et surtout intramusculaire) des autres voies locales d’administration, comme l’inhalation pulmonaire. Un affaissement du Cortisol urinaire avec des concentrations proches de zéro pourrait indiquer la nature de l’administration du glucocorticostéroïde. Néanmoins, des analyses complémentaires devraient encore être réalisées dans le but de confirmer et de généraliser cette observation. En particulier, sur un plus grand nombre de volontaires et surtout sur les échantillons provenant des IA et des autres applications locales.